Tuberculosis kills about 2 million people globally every year. A key defense against Mycobacterium tuberculosis (Mtb) infections is the production of nitric oxide (NO) by macrophages. Although NO controls Mtb growth, it rarely sterilizes the bacterium from the host, suggesting Mtb has mechanisms to resist NO toxicity. The Mtb proteasome is one such mechanism that is required for resistance to NO as well as causing death in mice. Thus, we are interested in targeting the proteasome and its associated factors for drug development. The proteasome is a multi-subunit, barrel shaped complex that degrades proteins. We found that the proteins Mpa and PafA are required for protein degradation: Mpa is thought to chaperone proteins into the proteasome core and PafA appears to be required for the attachment of a prokaryotic ubiquitin-like protein (Pup) onto substrates targeted for destruction. Pup represents the first known post-translational small protein modifier identified in any prokaryote. Little is known about how Pup is conjugated to its target substrates thus we propose to identify and characterize all proteins required for "pupylation" using genetic, biochemical and molecular biological techniques. In addition, we have recently discovered pupylation is reversible, thus we are in the process of characterizing the "depupylation" pathway. The elucidation of the Pup-proteasome sytem of Mtb will hopefully lay the foundation for the discovery and characterization of other posttranslational modification systems in all bacteria. Furthermore, these enzymes may represent new targets for the development of anti- tuberculosis drugs. PUBLIC HEALTH RELEVANCE: Tuberculosis therapy takes 6-9 months, a problem that leads to decreased compliance for taking antibiotics and increased chances of developing drug-resistance. The rise of extensively drug resistant (XDR) strains of M. tuberculosis has become a great public concern as it has recently made headlines in the popular press. Thus the new treatments for tuberculosis are needed now, and the pupylation pathway may provide new targets for drug development.